Type I diabetes, or IDDM, is an autoimmune disease caused by T cells that attack and destroy the insulin producing .beta.-cells located in the islets of the pancreas (Castano and Eisenbarth, 1990). The autoimmune process culminating in IDDM begins and progresses without symptoms. The disease surfaces clinically only when the cumulative loss of .beta.-cells exceeds the capacity of the residual .beta.-cells to supply insulin. Indeed, the collapse of glucose homeostasis and clinical IDDM is thought to occur only after 80-90% of the .beta.-cells have been inactivated by the immune system. Thus, patients who can be identified as suffering from IDDM are bound to be in an advanced stage of autoimmune destruction of their .beta.-cells. Moreover, diagnosis of incipient, preclinical diabetes by the detection of immunological markers of .beta.-cell autoimmunity can be made only after the onset of the autoimmune process. Therefore, the therapeutic quest is to find a safe, specific and effective way to turn off an autoimmune process that is already well underway.
The present inventors have examined this question before by studying the spontaneous diabetes developing in mice of the NOD strain, which is considered to be a faithful model of human IDDM (Castano and Eisenbarth, 1990). NOD mice develop insulitis around 4 weeks of age, which begins as a mild peri-islet infiltrate and progresses to severe intra-islet inflammation. Hyperglycemia, which attests to insulin insufficiency, begins in the females in our colony at about 14-17 weeks of age. By 35-40 weeks of age, almost all the female NOD mice have developed severe diabetes and most die in the absence of insulin treatment. Male NOD mice have a lower incidence of disease, for reasons that are not clear. The diabetes of NOD mice has been shown to be caused by autoimmune T cells (Bendelac et al., 1987).
T cell reactivity and autoantibodies to various antigens have been detected in human IDDM patients as well as in NOD mice (Elias, 1994), and it is not clear whether immunity to any single one of the possible target antigens is the primary cause of the disease. Beyond the question of causation is the question of therapy.
It has been demonstrated that the initiation of the autoimmune process in NOD mice can be prevented by subjecting the mice, before the onset of diabetes, to various manipulations such as restricted diet, viral infections, or non-specific stimulation of the immune system (Bowman et al., 1994). NOD diabetes is also preventable by induction of immunological tolerance in pre-diabetic mice to the antigen glutamic acid decarboxylase (GAD) (Kaufman et al., 1993; Tisch et al., 1993).
The present inventors have found previously that the diabetes of NOD mice can be prevented by T cell vaccination using T cells specific for the p277 peptide sequence of the human hsp60 molecule (Elias et al., 1991). This protein was formerly designated hsp65, but is now designated hsp60 in view of more accurate molecular weight information; by either designation, the proteins are the same.
The p277 peptide, being the epitope of the human hsp60 involved in IDDM and corresponding to positions 437-460 of the human hsp60 sequence, was first described in Israeli Patent Application No. 94241 of the present applicant and in Elias and Cohen, 1994, and has the following sequence:
Val-Leu-Gly-Gly-Gly-Cys-Ala-Leu-Leu-Arg-Cys-Ile-Pro-Ala-Leu-Asp-Ser-Leu-Thr -Pro-Ala-Asn-Glu-Asp (SEQ ID NO:1) PA1 (i) preparing a mononuclear cell fraction containing T cells from a blood sample obtained from said patient; PA1 (ii) adding to said mononuclear cell fraction an antigen selected from a peptide of sequence I of the invention; PA1 (iii) incubating said cell fraction in the presence of said antigen for a suitable period of time and under suitable culture conditions; PA1 (iv) adding a labeled nucleotide to the incubated cell culture of (iii) at a suitable time before the end of said incubation period to provide for the incorporation of said labeled nucleotide into the DNA of proliferating T cells; and PA1 (v) determining the amount of proliferating T cells by analysis of the amount of labeled nucleotide incorporated into said T cells. PA1 (i) an antigen selected from a peptide of sequence I; and PA1 (ii) a tagged antibody capable of recognizing the non-variable region of said anti-hsp60 antibodies to be detected. PA1 (i) an antigen selected from a peptide of sequence I; PA1 (ii) a suitable medium for culture of lymphocytes (T cells); and PA1 (iii) either a labeled nucleotide for the T cell proliferation test, or a cytokine, e.g., IFN-.gamma., IL-2, IL-4, IL-6, IL-10, TNF.alpha. or TGF.beta., assay kit, for the cytokine test.
Administration of the p277 peptide itself at the onset of insulitis was shown also to prevent the development of diabetes, probably by down-regulating the anti-p277 immunity that is essential for NOD diabetes (Elias et al., 1991; Israeli Patent Application No. 94241). Recent studies have indicated that the p277 peptide may also be used to reverse .beta.-cell autoimmunity that has progressed to an advanced stage (Elias and Cohen, 1994).
The laboratory of the present inventors has recently reported that a form of autoimmune diabetes can be induced in the C57BL/KsJ strain of mice by the administration of a very low dose of the .beta.-cell toxin streptozotocin (STZ) (Elias et al., 1994). Whereas the standard low dose of STZ of 40 mg/kg administered daily for 5 days usually induces clinical diabetes within 3 weeks, the administration of 30 mg/kg for 5 days induces clinical diabetes only after a lag period of about 3 months. This model of induced diabetes is marked by the appearance in the prodrome period of autoantibodies to insulin, anti-idiotypic antibodies to the insulin autoantibodies, and autoantibodies to hsp60. The mice also manifest spontaneous T-cell reactivity to hsp60 and to its p277 peptide (Elias et al., 1994). Thus, the lower than standard low dose of STZ appears to trigger an autoimmune process not unlike that observed in the spontaneous diabetes developing in NOD mice (Elias et al., 1990).
It is an object of the present invention to provide variants of peptide p277, such variants being useful for the diagnosis and treatment of IDDM.